...contacts that bring apposing membranes together and can wrap the membranes into stacked structures including... Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 2002...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies ...protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general...in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently...best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP...

...detect its co-localization with a membrane-localized mCherry or GFP protein. With this system, if the ...Lab monitored how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins...bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to...more OptoNBs, one against mCherry and one against GFP. Insertion of AsLOV2 into loop 1 always generated....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...proliferation and differentiation.  Since Ras is a membrane localized GTPase, the team fused a mCherry OptoNB...OptoNB-SOS fusion protein in cells that expressed membrane-localized mCherry. With darkness, SOS binds Ras...

...protein of interest. The green fluorescent protein (GFP) and mCherry, two of the most common tags currently...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...DREADD receptors need to be located on the plasma membrane to be activated by the DREADD actuator after it.... Although we found a larger proportion on the membrane in mice neurons, most of them were still cytoplasmic...limited expression of hM4Di‐mCherry at the plasma membrane in monkey neurons, we next hypothesized that a...improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino...resulted in abundant expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our ...

...switching, TRICK requires the following components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter...Plasmid 64542 or 64543) Before translation, both GFP and RFP are bound to the reporter, causing it to ...appear yellow. The act of translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the... contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus to bind cognate untranslated...verified that translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known...Oskar protein levels were negatively correlated with GFP intensity, showing that TRICK is an accurate readout...

...-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2]) of this membrane-embedding...pAAV.hSyn-FLEX.iGABASnFR.F102G pAAV-Syn-Archon1-KGC-GFP-ER2 pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2] pAAV-FLEX-tdTomato pAAV-GFAP104...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...37825  AAV8*, AAV9*  pAAV-CAG-GFP 50465  AAV8*, AAV9*  pAAV-hSyn-EGFP *Also coming in our new 20 ...GABA sensor (pAAV.hSyn-FLEX.iGABASnFR) has good membrane localization and brightness. The F102G variant...pAAV-GFAP104-mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP,and pAAV-hSyn-mCherry at Addgene. Viral vectors coming...IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR IRES EGFP Chimeric...

...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing plasma membrane-targeted SuperPLDx30...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or .... The Chow Lab has also generated a variety of membrane-recruitment optogenetic tools for manipulating...

...sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when the receptor ...on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor...performed with a probe surrounded by a semipermeable membrane, which is inserted into the region of the brain...passive diffusion of DA across the semipermeable membrane from the extracellular fluid into the perfused...

...protein) associates with autophagosomal membranes, so tagging it with GFP allows for tracking the localization...Autophagosomes are relatively easy to identify: double-membraned structures containing undigested cytoplasmic contents...electron microscopy analysis is time consuming. GFP-tagged Atg8p or LC3: ATG8 is part of a group of genes...autophagosomes and autophagic bodies in yeast. A GFP-tagged version of LC3, the mammalian Atg8p homolog...pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). They are ...Fluorescent Protein Oligomerization Seeing Red: Simple GFP Photoconversion Light Sheet Fluorescence Microscopy...

...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...regulation. To create iGluSnFR, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic...increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing ...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In ...generation of iGluSnFR, the Looger lab swapped eGFP for sfGFP to create SF-iGluSnFR. This improved variant.... PDGFR is a C-terminal fusion to the PDGFR transmembrane domain in the mammalian expression pMinDisplay...glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane domain, followed by a modified form of the cytosolic...

...Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy biosensor contains mRFP and GFP fused to the...flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched...decrease in red puncta. Find the mRFP-GFP-LC3 plasmid at Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe ...autophagy biosensor is the GFP-LC3-RFP-LC3ΔG probe. It’s similar to the mRFP-GFP-LC3 Tandem Fluorescent Protein...molecule of GFP-tagged LC3 for every one molecule of RFP-tagged LC3∆G. Like the unmodified LC3, GFP-LC3 is ...Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence...variant, DsRed.T3, that’s connected to a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). In the absence...

...al. observed some GFP aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility...scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. Although traditional...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power...finding that plasma membrane-targeted CAAX-SunTag was 18-fold brighter than sfGFP! The high SunTag signal... copies of the short epitope GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed...physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they ...they examined the effects of mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria...

... from the default GFP (green) to RFP, CFP, or YFP fluorescence. In Brainbow 2.1, GFP is tagged with a ... cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic RFP in ... the fluorophores RFP (red), YFP (yellow), and membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange...and activate fluorescence of the downstream OFP, GFP, or RFP. Cells that express the construct but do ...control of Flp recombinase. The XFPs in Flybow are membrane-tethered, making this system useful for visualization...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically low...

...to endogenously tag a wide variety of genes with GFP. These plasmids are available through Addgene, and...is the addition of a useful coding sequence (i.e. GFP) into a gene while leaving other features of the ...we show an example gene targeted for tagging with GFP at the C terminus, resulting in one edited allele...allele, or gene copy. The coding sequence for GFP is transcribed and translated along with the gene, producing...shown in the example above, we inserted a linker and GFP tag at the end of the last exon of the gene to ensure...matching the gene of interest, a linker, and the GFP tag sequence to be inserted. Created with BioRender.com...Cas9 makes at the target site. The whole segment—a GFP tag and 1kb of homologous DNA flanking both sides...

...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same pattern...endoplasmic reticulum (ER), vacuole or cellular membranes. Examples include blue flame mitochondria-targeted...an organelle-specific targeting signal or a transmembrane domain, biosensors can be targeted to a particular...

...hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~...spontaneously assemble into particles at the cell membrane. Mature Nanoblades then bud into the cell culture... where they can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote...

...GTPase in division) GFP-Mff (outer membrane protein) Late Endosome mCherry-Rab7A GFP-Rab7A     ...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...and plasma membrane reporters as well as secretory proteins or proteins targeted to membrane sub-domains...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...protein Knock-off, is made up of three components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter...these dCas9s with one of three fluorescent proteins (GFP, BFP and mCherry), the authors were able to visualize...

... AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral vectors ...supernatant, the viral particles associate with the cell membrane and are taken into the cell via endocytosis. In...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...

...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ... your research, check out this post! R Bodies: Membrane-Rupturing Microscopic Tools In this post, Jessica...also showed that R-bodies can rupture E. coli membranes. Read the full post for potential applications...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness... There are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made...examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and...its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles a cylinder...2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins are made by ... a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps... al., 2018). GFP is not tolerated in my fusion It is standard practice to attach the GFP to the N- or ...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the...enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity...relies on several observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly...extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished by knocking down...